Microparticles have been studied extensively by the pharmaceutical industry, drug delivery companies and academic research groups for more than three decades now with significant success. It has been shown that microparticles are very useful carriers for active compounds in the broadest sense. They can be loaded with drugs, diagnostic agents, vaccines, or genetic material and administered to deliver these active materials to an organism or to specific cells or tissues, optionally providing controlled release or specific binding to targeted structures. In some applications, microparticles have proven useful even when they do not contain active compounds, e.g. in the controlled embolization of blood vessels. As diagnostic carriers or as transfection systems, they may not have to be administered to e.g. a plant, an animal or to a human organism, but can be used in vitro to produce the desired effects.
Microparticles have been defined and classified in various different ways depending on their specific structure, size, or composition. As used herein, microparticles are broadly defined as micro- or nanoscale particles which are typically composed of solid or semi-solid materials and which are capable of carrying an active compound. Typically, the weight-average diameter of such microparticles ranges from approximately 10 nm and approximately 500 μm. More preferably, the average particle diameter is between about 50 nm and 100 μm. Several types of microparticle structures can be prepared according to the invention. These include substantially homogenous structures, such as nano- and microspheres, nano- and microparticles, solid lipid nanoparticles, and the like. They also include particles with a structure comprising an inner core and an outer coating, such as nano- and microcapsules. They further include particles formed by the colloidal association of small molecules, especially of amphiphilic molecules, or other complex structures, such as liposomes, lipoplexes, lipid complexes, and lipospheres.
Microparticles are capable of carrying one or more active compounds. For instance, an active compound may be more or less homogeneously dispersed within the microparticles or within the microparticle cores. Alternatively, it may be located within the microparticle shell or coating. In complex particles, such as liposomes or lipospheres, the active compound may be associated with certain hydrophilic or lipophilic regions formed by the associated amphiphilic molecules contained in such particles, depending on the hydrophilicity or lipophilicity of the compound.
Pharmaceutical products with drug-containing microparticles have been developed which improve certain therapies with injectable drugs. For instance, parenteral depot formulations based on microparticles are appreciated by patients and health care providers as they allow a drug to be administered at a greatly reduced dosing frequency, such as once a month instead of daily. An example for a successful product line based on this concept is Lupron® depot, containing the drug leuprolide, which is released over a period of 1 to 4 months depending on the specific product and formulation. In this product, the microparticles are primarily composed of biodegradable polymers. Other examples for the successfully commercialized applications of microparticles are the products AmBisome®, DaunoXome®, Doxil®, Amphotec®, and Abelcet®, which are based on liposomes or similar lipid-based microparticulate structures or complexes.
Several methods for the preparation of microparticles have been described, the majority of which involve the formation of the particles in a fluid or liquid carrier. For instance, polymeric microparticles may be formed by chemical, physicochemical or physical processes involving interfacial polymerization, dispersed-phase crosslinking, complex coacervation, coacervation, thermal denaturation, salting-out, solvent evaporation, hot-melt techniques, solvent removal, solvent extraction, or phase separation. A good description and summary of many of the microparticle formation processes used today is found in the Encyclopedia of Controlled Drug Delivery, ed. by E. Mathiowitz, Wiley-Interscience, vol. 2, article “Microencapsulation”, which is incorporated herein by reference to describe suitable microparticle preparation processes.
Typically, lipid-based microparticulate systems such as liposomes are formed in liquids, most often in aqueous liquids. Among the principal processes used for their preparation are lipid film hydration, dehydration-rehydration, reversed-phase evaporation, organic solution injection, and detergent removal. Very often, the crude structures thus obtained are further processed to adjust the particle size or to increase the load of active compound. Such processes may include membrane extrusion, high pressure homogenization, treatment with ultrasound, dehydration-rehydration, freeze-thaw cycles and the like. The most common methods to prepare liposomes and other lipid-based micro- and nanostructures are reviewed in the Encyclopedia of Controlled Drug Delivery, ed. by E. Mathiowitz, Wiley-Interscience, vol. 1, article “Liposomes”, which is incorporated herein by reference to describe suitable microparticle preparation processes.
Considering the wealth of information available on methods for the preparation of microparticles, comparatively little attention has been paid to any processing steps following the formation of the particles which are nevertheless needed to obtain a product which is suitable and acceptable for pharmaceutical and diagnostic uses. In particular, the purification aspect has not been solved in a satisfactory way in many of the known preparation methods.
The purification of the particles after their formation is important for a number of reasons. For instance, a more or less significant amount of free active compound may be present in the liquid in which the microparticles were formed, as most preparation methods lead to loading rates of clearly less than 100%. Unbound or unincorporated active compound is mostly undesired because it behaves differently from incorporated material in terms of pharmacokinetics, toxicity, effectiveness, or stability. It is therefore highly desirable to remove unbound active material from the microparticle dispersion prior to its use. The same is true for other substances, which may include unincorporated fractions of microparticle components or excipients, but also substances which were needed in the process of microparticle formation only, or which were produced during that process. Examples for such impurities whose removal from the microparticle dispersion may be desired include crosslinking reagents, initiators, reaction product, degradation products, stabilizers, surfactants, detergents, thickeners, solvents, cosolvents, substances for adjusting the pH, osmotic pressure, ionic strength, or zeta potential, etc. In a further aspect, it may be useful to remove microparticles that are outside the desired size range of the particles, as very small or very large microparticles—relative to the target diameter for a specific application—may behave quite differently from proper sized particles.
In the prior art, a few methods have been described to separate undesired substances or particles from microparticles in dispersions. For instance, microparticles may simply be washed on a filter with a washing liquid. Such a method is e.g. described in U.S. Pat. No. 4,652,441, which is incorporated herein by reference. Another known option is centrifugation, which is used to obtain a concentrated microparticle “pellet” and a supernatant which is subsequently removed by decantation. Typically, the process is repeated several times to obtain an acceptable level of purity. Centrifugation, however, has several disadvantages. First, it exerts some substantial mechanical force on the microparticles, which may damage their structure or lead to the formation of aggregates that cannot easily be dispersed again. If the microparticles are liposomes, centrifugation may prove destructive altogether. On the other hand, if the microparticles are very small, i.e. in the submicron range, or even below about 200 nm in diameter, high rotation speeds have to be applied in order to achieve particle sedimentation, making this process costly, energy-consuming, and not very efficient. It is also difficult to set up centrifugation as a continuous or semi-continuous process, or to scale it up to industrial throughput requirements.
These concerns are especially valid when the purification of microparticles must be conducted under aseptic conditions. This is often the case since one of the preferred uses of microparticles is in the parenteral administration of active compounds. Any product for parenteral use must be sterile, and while many conventional injectables can be sterilized after their preparation, this is not often an option for microparticle formulations. Injectable solutions are most often sterilized in their vials by autoclaving. Sometimes it is also possible to sterilize the final product by gamma- or e-beam radiation.
If these sterilization methods cannot be used due to the insufficient stability of the formulation, the solution may be filtrated to remove any microbial contaminants and subsequently filled into the vials under aseptic conditions. However, bacteria cannot reliably be separated from microparticles unless the particles are very small, such as smaller than 0.2 μm in diameter. Many types of microparticles are larger than this. Furthermore, microparticles are often loaded with highly sensitive compounds, such as peptides, proteins, or nucleic acids, which are not stable enough to allow heat sterilization.
Partly in response to some of the needs mentioned above, it has been suggested to purify microparticles by methods involving filtration or dialysis, which are comparatively gentle methods. For instance, U.S. Pat. No. 5,069,936 discloses the preparation of protein microspheres with subsequent purification by centrifugation and washing, gradient centrifugation, but also by dialysis, gel-filtration, electrophoresis, column chromatography, thin-layer chromatography, hollow-fiber ultrafiltration, and tangential flow filtration. According to U.S. Pat. Nos. 5,962,566, 5,100,591, 5,069,936, and 5,947,689, microparticles of various types can be purified by tangential ultrafiltration or diafiltration. However, they do not teach how this method can be carried out aseptically at a large manufacturing scale. Some more advanced solutions to these problems are disclosed in U.S. Pat. No. 6,264,988, even though this document addresses a very specific and rather complicated process for the manufacture of fibrinogen-coated cross-linked albumin microparticles. There still remains the need for improved methods to prepare purified microparticles, i.e. for methods which are efficient, easily scalable to large batch volumes, allow a high degree of continuous processing, are easily conducted under aseptic conditions, and allow the gentle processing of highly sensitive and instable materials. There also is a need for improved apparatuses, processing lines and assemblies which can be used for conducting such methods.
It is therefore an object of the invention to provide an improved method for preparing microparticles. More particularly, an object is to provide an aseptic method for the preparation of purified, drug-loaded microparticles. In another aspect, it is an object of the invention to provide a method for the preparation of purified, drug-loaded microparticles which is easy to scale-up to large production volumes. Furthermore, it is an object of the invention to provide a method which allows a high degree of continuous processing. In a further aspect, it is an object of the invention to provide apparatuses and assemblies of processing equipment which can be used to prepare and purify microparticles. Yet in another aspect, it is an object to provide purified microparticles obtained by the method of the invention, and pharmaceutical compositions comprising such microparticles.